**1. Introduction**

[37] Raithel, D., Recurrent carotid disease: optimum technique for redo surgery. J Endo‐

[38] Frericks, H., et al., Carotid recurrent stenosis and risk of ipsilateral stroke: a system‐

[39] AbuRahma, A.F., et al., Redo carotid endarterectomy versus primary carotid endar‐

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[41] Setacci, F., et al., Carotid restenosis after endarterectomy and stenting: a critical is‐

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124 Carotid Artery Disease - From Bench to Bedside and Beyond

#### **1.1. Carotid stenosis and atheromatous process**

Carotid artery stenosis due to atherosclerosis is a major complication of hyperlipidemia, diabetes mellitus and hypertension. Moreover, the extent of carotid intima media thickness is a measure of atheromatosis and therefore of cardiovascular disease (CVD).

The effect of cholesterol in the process of atheromatosis is now well established. High levels of total cholesterol, as well as of low-density lipoprotein (LDL), very low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), lipoprotein a (Lp-α), and triglycerides, coupled with decreased levels of high-density lipoprotein (HDL) are responsible for the creation of atheromatous plaques [1-3]. Of the above factors, LDL cholesterol, and especially the oxidized LDL is considered as the most important contributor of atheromatosis [4].

The atheromatous process is completed in the following three stages:

**1.** In the first stage, LDL cholesterol enters the vessel wall, binds to gluxosaminoglucanes, which are part of the extracellulat matrix of the intima. This binding is facilitated by apolipoprotein B-100 (ApoΒ–100). The accumulation of LDL in the vessel wall contributes to the formation of fatty strikes. Following LDL adhesion in the vessel wall, it undergoes oxidation by free radicals produced locally, the molecule is altered and chemokines are produced by adjacent vessel wall cells, such as MCP – 1, together with growth factors, which are responsible for the accumulation of monocytes and macrophages. The latter,

cause further oxidation of LDL, resulting in negative charge, recognition by scavenger receptors on macrophage membrane and increased uptake of LDL inside the macrophag‐ es, as these receptors are not inhibited by increased intracellular concentration of choles‐ terol. The final result is an enormous accumulation of LDL in the macrophages, which are transformed to foamy cells. These cells represent the first step in the atheromatous process [5] (figure 1).

**3.** In the third stage, that of the complicated lesion, the rapture of the fibrous capsid of the atheromatous plaque leads to massive evacuation of the cholesterol reservoir. The artery may occult due to the accumulation of platelets and clotting, leading to infarction (figure

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Avoiding the formation and the instability of the atheromatous plaque is top priority for patients at risk for cardiovascular events. Statins may contribute towards this direction [6,7]

**Figure 2.** Advanced atheromatous plaque causing arterial lumen occlusion of 70% (adopted from Durrington & Sin‐

**Figure 3.** The point of the atheromatous plaque which active enlargement is occurring. Formation of new foamy cells, increased cholesterol uptake and increased instability of the plaque (adopted from Durrington & Sinderman, 2002).

derman, 2002).

4). If not so, then the plaque will be further enlarged [5].

**2.** During the second stage, the atheromatous plaque is formed. Foamy cells produce growth factors and together with oxidized LDL result to the attraction of smooth muscle cells. The latter, are then differentiated to fibroblasts and start producing collagen. This collagen covers foamy cells, which either are destroyed or are forced to apoptosis. The final result is the formation of a pool of extracellular cholesterol trapped under a fibrous capsid (figure 2). The part which is close to the yet intact vessel wall is the active one of the plaque, where the foamy cells are produced. As the plaque extents to the inner layers of the vessel wall, the point of foamy cell formation becomes instable and may cause rapture of the plaque [5] (figure 3).

**Figure 1.** Atherogenesis. Fatty strikes are characterized by macrophages containing an excess of lipids (foamy cells). Foamy cells are derived by blood monocytes which are attracted to vessel intima and start phagocytosing lipoproteins, such as oxidized LDL. The convertion of fatty strike to atheroma depends on proliferation and differentiation of smooth muscle cells to fibroblasts. The latter produce collagen resulting in intima thickening. As the lesion extents further, foamy cells are destroyed releasing large amounts of cholesterol trapped in a fibrous capsid. The active site of atheroma is the point which is adjacent to normal endothelium, where foamy cells are formed (adopted from Durring‐ ton & Sinderman, 2002).

**3.** In the third stage, that of the complicated lesion, the rapture of the fibrous capsid of the atheromatous plaque leads to massive evacuation of the cholesterol reservoir. The artery may occult due to the accumulation of platelets and clotting, leading to infarction (figure 4). If not so, then the plaque will be further enlarged [5].

cause further oxidation of LDL, resulting in negative charge, recognition by scavenger receptors on macrophage membrane and increased uptake of LDL inside the macrophag‐ es, as these receptors are not inhibited by increased intracellular concentration of choles‐ terol. The final result is an enormous accumulation of LDL in the macrophages, which are transformed to foamy cells. These cells represent the first step in the atheromatous process

**2.** During the second stage, the atheromatous plaque is formed. Foamy cells produce growth factors and together with oxidized LDL result to the attraction of smooth muscle cells. The latter, are then differentiated to fibroblasts and start producing collagen. This collagen covers foamy cells, which either are destroyed or are forced to apoptosis. The final result is the formation of a pool of extracellular cholesterol trapped under a fibrous capsid (figure 2). The part which is close to the yet intact vessel wall is the active one of the plaque, where the foamy cells are produced. As the plaque extents to the inner layers of the vessel wall, the point of foamy cell formation becomes instable and may cause rapture of the plaque

**Figure 1.** Atherogenesis. Fatty strikes are characterized by macrophages containing an excess of lipids (foamy cells). Foamy cells are derived by blood monocytes which are attracted to vessel intima and start phagocytosing lipoproteins, such as oxidized LDL. The convertion of fatty strike to atheroma depends on proliferation and differentiation of smooth muscle cells to fibroblasts. The latter produce collagen resulting in intima thickening. As the lesion extents further, foamy cells are destroyed releasing large amounts of cholesterol trapped in a fibrous capsid. The active site of atheroma is the point which is adjacent to normal endothelium, where foamy cells are formed (adopted from Durring‐

[5] (figure 1).

126 Carotid Artery Disease - From Bench to Bedside and Beyond

[5] (figure 3).

ton & Sinderman, 2002).

Avoiding the formation and the instability of the atheromatous plaque is top priority for patients at risk for cardiovascular events. Statins may contribute towards this direction [6,7]

**Figure 2.** Advanced atheromatous plaque causing arterial lumen occlusion of 70% (adopted from Durrington & Sin‐ derman, 2002).

**Figure 3.** The point of the atheromatous plaque which active enlargement is occurring. Formation of new foamy cells, increased cholesterol uptake and increased instability of the plaque (adopted from Durrington & Sinderman, 2002).

secretory activity of endothelial cells [15], inhibiting the nitric oxide-mediated vasodilatation through reduction of the expression of endothelial nitric-oxide synthase (eNOS), inducing the expression of adhesion molecules on the endothelium thus mediating the adhesion of mono‐ cytes to intima [15], and inducing the expression of inflammatory cytokines [16]. Indeed, oxLDL is a potent inducer of inflammation [17], contributing to the chronic inflammatory

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The 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors, or statins, reduce total cholesterol (TC), LDL cholesterol, apolipoprotein B (apoB), and, to a lesser degree, triglycerides and lipoprotein a (Lp-a). Statins also have pleiotropic effects [19], such as the modulation of inflammatory molecules and monocyte maturation and differentiation [19], the suppression of smooth muscle-cells migration and proliferation [19], the reduction of the monocyte adhesion to the endothelium [20], the restoration of the impaired endothelium-dependent vessel wall relaxation [21], and the modification of cell-mediated LDL oxidation [22,23]. All of the above mechanisms contribute to the reversion of atheromatosis. Undeniably, statins reduce the incidence of coronary events and are a cornerstone in the primary and secondary preven‐ tion of CHD [24]. Previous studies have detected some efficacy in reducing the circulating oxLDL levels, but whether this effect is due to the reduction of LDL or is an independent, pleiotropic phenomenon remains a matter of controversy [25,26]. Furthermore, little is known

The aim of the present study was to evaluate the efficacy of atorvastatin in reducing stenosis, to investigate the effect on oxLDL and to search for possible associations of oxLDL modification with changes of stenosis in patients managed conservatively and in pre-treated with percuta‐ neous catheter interventional procedures patients with carotid atheromatosis. We hypothesise that atorvastatin therapy will confer remission of oxLDL levels in vivo and this will be

Between January 2005 and February 2008 a total of 100 patients were randomly selected from the lipid clinic and the carotid angioplasty clinic of a large tertiary hospital in Athens for inclusion in the study. Informed consent was obtained from each patient at recruitment according to our institutional policies. Eligible were patients with carotid artery stenosis from various causes and with a range of predisposing factors. Exclusion criteria included: acute cardiovascular disease, severe or unstable angina pectoris, clinically evident cardiac failure, severe arrhythmias, recent surgical procedures, inflammatory diseases, active liver disease or liver impairment, excessive alcohol consumption (>4 units/day) or history of alcohol abuse, known allergic reaction to statins, poorly controlled diabetes mellitus as defined by a haemo‐ globin A1c (HbA1c) level of >7mg/dl, uncontrolled hypertension indicated by systolic blood pressure (SBP) >140mmHg and/or diastolic pressure >85mmHg, history of deep vein throm‐

about the definite clinical benefit of such oxidative marker reduction.

associated with significant reduction of carotid artery stenosis.

**2. Patients and methods**

process which results to atherosclerosis [18].

**1.3. Statins**

**Figure 4.** A raptured ahteromatous plaque, in which the cholesterol reservoir has evacuated itself under the fibrous capsid. A clot has in the endothelial surface at the site of rapture is completely occulting the lumen (adopted from Durrington & Sinderman, 2002).

### **1.2. Oxidised LDL**

Oxidized low density lipoprotein LDL (oxLDL) cholesterol in humans is found mainly in two types:


Oxidized LDL is produced following oxidation of LDL by free radicals and other oxidadive factors, a procedure called oxidative stress. The circulating oxidized LDL is the measurable fraction of oxidized LDL in plasma. Oxidised LDL is a key element of the pathway leading to the formation of the atheromatous plaque and has been extensively studied both as a marker of atheromatosis and as a possible target of therapeutic intervention. Circulating oxLDL is considered a risk marker for atherosclerosis [8] and coronary heart disease (CHD) [8-10]. Increased oxLDL levels in circulation and the vessel wall are associated with endothelial dysfunction [11] in such patients [9,10,12], contributing to atheromatous plaque instability [9].

Oxidative modification of LDL leads to rapid focal accumulation in macrophages [13], which is the first step in atheromatous process. The increased retention time of LDL in the intima offers enhanced probability to be oxidized by free radicals produced by endothelium, smooth muscle cells or macrophages [14]. Oxidized LDL then acts chemotactic for monocytes and smooth muscle cells through binding to scavenger receptors [15], leading to the formation of foam cells. Oxidized LDL is also capable to elicit endothelial dysfunction by altering the secretory activity of endothelial cells [15], inhibiting the nitric oxide-mediated vasodilatation through reduction of the expression of endothelial nitric-oxide synthase (eNOS), inducing the expression of adhesion molecules on the endothelium thus mediating the adhesion of mono‐ cytes to intima [15], and inducing the expression of inflammatory cytokines [16]. Indeed, oxLDL is a potent inducer of inflammation [17], contributing to the chronic inflammatory process which results to atherosclerosis [18].

#### **1.3. Statins**

**Figure 4.** A raptured ahteromatous plaque, in which the cholesterol reservoir has evacuated itself under the fibrous capsid. A clot has in the endothelial surface at the site of rapture is completely occulting the lumen (adopted from

Oxidized low density lipoprotein LDL (oxLDL) cholesterol in humans is found mainly in two

Oxidized LDL is produced following oxidation of LDL by free radicals and other oxidadive factors, a procedure called oxidative stress. The circulating oxidized LDL is the measurable fraction of oxidized LDL in plasma. Oxidised LDL is a key element of the pathway leading to the formation of the atheromatous plaque and has been extensively studied both as a marker of atheromatosis and as a possible target of therapeutic intervention. Circulating oxLDL is considered a risk marker for atherosclerosis [8] and coronary heart disease (CHD) [8-10]. Increased oxLDL levels in circulation and the vessel wall are associated with endothelial dysfunction [11] in such patients [9,10,12], contributing to atheromatous plaque instability [9].

Oxidative modification of LDL leads to rapid focal accumulation in macrophages [13], which is the first step in atheromatous process. The increased retention time of LDL in the intima offers enhanced probability to be oxidized by free radicals produced by endothelium, smooth muscle cells or macrophages [14]. Oxidized LDL then acts chemotactic for monocytes and smooth muscle cells through binding to scavenger receptors [15], leading to the formation of foam cells. Oxidized LDL is also capable to elicit endothelial dysfunction by altering the

**a.** conjugated form, attached to the atheromatous plaque and

Durrington & Sinderman, 2002).

**b.** circulating form found in serum.

128 Carotid Artery Disease - From Bench to Bedside and Beyond

**1.2. Oxidised LDL**

types:

The 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors, or statins, reduce total cholesterol (TC), LDL cholesterol, apolipoprotein B (apoB), and, to a lesser degree, triglycerides and lipoprotein a (Lp-a). Statins also have pleiotropic effects [19], such as the modulation of inflammatory molecules and monocyte maturation and differentiation [19], the suppression of smooth muscle-cells migration and proliferation [19], the reduction of the monocyte adhesion to the endothelium [20], the restoration of the impaired endothelium-dependent vessel wall relaxation [21], and the modification of cell-mediated LDL oxidation [22,23]. All of the above mechanisms contribute to the reversion of atheromatosis. Undeniably, statins reduce the incidence of coronary events and are a cornerstone in the primary and secondary preven‐ tion of CHD [24]. Previous studies have detected some efficacy in reducing the circulating oxLDL levels, but whether this effect is due to the reduction of LDL or is an independent, pleiotropic phenomenon remains a matter of controversy [25,26]. Furthermore, little is known about the definite clinical benefit of such oxidative marker reduction.

The aim of the present study was to evaluate the efficacy of atorvastatin in reducing stenosis, to investigate the effect on oxLDL and to search for possible associations of oxLDL modification with changes of stenosis in patients managed conservatively and in pre-treated with percuta‐ neous catheter interventional procedures patients with carotid atheromatosis. We hypothesise that atorvastatin therapy will confer remission of oxLDL levels in vivo and this will be associated with significant reduction of carotid artery stenosis.
